MLKL overexpression leads to Ca2+ and metabolic dyshomeostasis in a neuronal cell model.

• MLKL overexpression in a neuronal cell model marginally affects viability. • MLKL regulates cytosolic Ca²⁺ clearance and increases Ca²⁺ release from the endoplasmic reticulum in Neuro-2a cells. • Transient Ca²⁺ elevations in MLKL-overexpressing Neuro-2a cells lead to higher cytosolic lactate levels. The necroptotic effector molecule MLKL accumulates in neurons over the lifespan of mice, and its downregulation has the potential to improve cognition through neuroinflammation, and changes in the abundance of synaptic proteins and enzymes in the central nervous system. Notwithstanding, direct evidence of cell-autonomous effects of MLKL expression on neuronal physiology and metabolism are lacking. Here, we tested whether the overexpression of MLKL in the absence of cell death in the neuronal cell line Neuro-2a recapitulates some of the hallmarks of aging at the cellular level. Using genetically-encoded fluorescent biosensors, we monitored the cytosolic and mitochondrial Ca²⁺ levels, along with the cytosolic concentrations of several metabolites involved in energy metabolism (lactate, glucose, ATP) and oxidative stress (oxidized/reduced glutathione). We found that MLKL overexpression marginally decreased cell viability, however, it led to reduced cytosolic and mitochondrial Ca²⁺ elevations in response to Ca²⁺ influx from the extracellular space. On the contrary, Ca²⁺ signals were elevated after mobilizing Ca²⁺ from the endoplasmic reticulum. Transient elevations in cytosolic Ca²⁺, mimicking neuronal stimulation, lead to higher lactate levels and lower glucose concentrations in Neuro-2a cells when overexpressing MLKL, which suggest enhanced neuronal glycolysis. Despite these alterations, energy levels and glutathione redox state in the cell bodies remained largely preserved after inducing MLKL overexpression for 24–48 h. Taken together, our proof-of-concept experiments are consistent with the hypothesis that MLKL overexpression in the absence of cell death contributes to both Ca²⁺ and metabolic dyshomeostasis, which are cellular hallmarks of brain aging. [Display omitted] [ABSTRACT FROM AUTHOR]